Discovery and Characterization of 1 H-1,2,3-Triazole Derivatives as Novel Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy

Article abstract of DOI:10.1021/acs.jmedchem.9b01269

The prostanoid EP4 receptor is one of the key receptors associated with inflammatory mediator PGE₂-elicited immunosuppression in the tumor microenvironment. Blockade of EP4 signaling to enhance immunity-mediated tumor elimination has recently emerged as a promising strategy for cancer immunotherapy. In our efforts to discover novel subtype-selective EP4 antagonists, we designed and synthesized a class of 1H-1,2,3-triazole-based ligands that display low nanomolar antagonism activity toward the human EP4 receptor and excellent subtype selectivity. The most promising compound 59 exhibits single-digit nanomolar potency in the EP4 calcium flux and cAMP-response element reporter assays and effectively suppresses the expression of multiple immunosuppression-related genes in macrophage cells. On the basis of its favorable ADMET properties, compound 59 was chosen for further in vivo biological evaluation. Oral administration of compound 59 significantly inhibited tumor growth in the mouse CT26 colon carcinoma model accompanied by enhanced infiltration of cytotoxic T lymphocytes in the tumor tissue.

Full text of DOI:10.1021/acs.jmedchem.9b01269

Subscriber access provided by UNIV TWENTE
Article
Discovery and Characterization of 1H-1,2,3-Triazole Derivatives as
Novel Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy
Jun-Jie Yang, Wei-Wei Yu, Long-Long Hu, Wen-Juan Liu, Xian-Hua Lin, Wei Wang, qiansen
zhang, Peili Wang, Shuo-Wen Tang, xin wang, Mingyao Liu, Weiqiang Lu, and Hankun Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01269 • Publication Date (Web): 19 Dec 2019
Downloaded from pubs.acs.org on December 22, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery and Characterization of 1H-1,2,3-Triazole
Derivatives as Novel Prostanoid EP4 Receptor
Antagonists for Cancer Immunotherapy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
†
Jun-Jie Yang , Wei-Wei Yu , Long-Long Hu, Wen-Juan Liu, Xian-Hua Lin, Wei Wang,
Qiansen Zhang, Pei-Li Wang, Shuo-Wen Tang, Xin Wang, Mingyao Liu, Weiqiang Lu*,
and Han-Kun Zhang*
Drug Discovery Unit, Shanghai Key Laboratory of Regulatory Biology, Institute of
Biomedical Sciences and School of Life Sciences, East China Normal University, 500
Dongchuan Road, Shanghai 200241, China
ABSTRACT
The prostanoid EP4 receptor is one of the key receptors associated with
inflammatory mediator PGE -elicited immunosuppression in the tumor microenvironment.
2
Blockade of EP4 signaling to enhance immunity-mediated tumor elimination has recently
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
emerged as a promising strategy for cancer immunotherapy. In our efforts to discover
novel subtype-selective EP4 antagonists, we designed and synthesized a class of 1H-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,2,3-triazole-based ligands that display low nanomolar antagonism activity towards the
human EP4 receptor and excellent subtype selectivity. The most promising compound 59
exhibits single-digit nanomolar potency in the EP4 calcium flux and CRE reporter assays,
and effectively suppresses the expression of multiple immunosuppression-related genes
in macrophage cells. On the basis of its favorable ADMET properties, compound 59 was
chosen for further in vivo biological evaluation. Oral administration of compound 59
significantly inhibited tumor growth in the mouse CT26 colon carcinoma model
accompanied by enhanced infiltration of cytotoxic T lymphocytes in tumor tissue.
ACS Paragon Plus Environment
2

Page 3 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Prostanoids are a family of endogenous chemical mediators expressed in various
human tissues and fluids, which are biosynthesized from arachidonic acid through the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
activity of cyclooxygenases (COX1 and COX2), peroxidases, and downstream tissue-
specific synthases. These bioactive lipids generally contain twenty carbons including a
five-membered carbon ring and play essential roles in maintaining body homeostasis.
Among them, prostaglandin E (PGE ) is the most abundant prostaglandin within the
2
2
human body and has been widely implicated in numerous physiological and
pathophysiological processes, including inflammation, pain, atherosclerosis, renal
disease, osteoporosis, and cancer. Recently, a growing body of evidence supports
2
chronic inflammation as a major factor promoting cancer development. As an important
pro-inflammatory mediator, PGE is heavily involved in modulating tumor progression
2
through multiple signaling pathways regulating angiogenesis and immunosuppression,
and is often correlated with a poor prognosis in cancer patients.3, 4 In contrast, 15-
hydroxyprostaglandin dehydrogenase (15-PGDH), a primary PGE -degrading enzyme
2
highly expressed in normal tissues, is found to be ubiquitously downregulated in various
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
human cancers. Upregulation of COX2 expression in tumor cells and myeloid cells can
result in elevated levels of PGE , which diffuses into the tumor microenvironment and
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
subsequently binds to a cluster of four G protein-coupled receptors (GPCRs), referred to
as EP receptors (EP1-4). Among the four receptors, the EP4 receptor is mainly expressed
in myeloid cells in the tumor microenvironment, and is the major contributor to the PGE2-
elicited immunosuppressive activity that promotes tumor development and progression.6-8
Upon ligand binding, G -coupled EP4 receptor stimulates the intracellular production of
S
9
cyclic AMP (cAMP) and the consequent activation of protein kinase A (PKA). The cAMP
pathway is thought to be the primary signal for EP4 receptor mediated
6
immunosuppression. Previous studies have demonstrated that selective deletion of EP4
receptor in myeloid cells markedly inhibited the development and progression of
colorectal cancer in ApcMin/+ mice.10 Meanwhile, pharmacologically blocking the EP4
receptor could induce effective anti-tumor immune responses in vivo by reducing
11
intratumoral immunosuppressive myeloid cells. So far, numerous preclinical studies and
clinical trials have indicated that long-term administration of traditional nonsteroidal anti-
inflammatory drugs (NSAIDs) or selective COX2 inhibitors is beneficial for cancer
ACS Paragon Plus Environment
4

Page 5 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
chemoprevention,12 however, these agents may cause severe or even life-threatening
gastrointestinal (GI) bleeding and ulcers in some patients, and increase the risk of heart
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
attacks, strokes, and heart-related deaths especially for long-term medication. Besides,
under physiological conditions, PGE is responsible for a wide range of homeostasis in
2
mammals, and NSAIDs used to decrease the production of PGE may be associated with
2
a diverse range of pathological conditions. A growing body of studies have indicated that
a selective EP4 antagonist could be an effective alternative with better GI tolerability
compared to NSAIDs.14 Furthermore, a preferable cardiovascular safety profile can be
envisaged with the proper use of selective EP4 antagonists because they do not interfere
with the biosynthesis of other prostanoids such as PGI .15 Given these advantages of
2
EP4 antagonists and the recent demonstration of the crystal structure of human EP4
receptor in complex with an antagonist,16 selective inhibition of PGE /EP4 signaling
2
pathway represents an attractive strategy for cancer immunotherapy.
To date, a number of structurally diverse EP4 antagonists have been identified and
developed for clinical studies in multiple diseases, such as pain, inflammation disorders,
17
and cancer (Figure 1). Compound 2 (Grapiprant), the first and only EP4 antagonist on
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the market from Aratana Therapeutics, was approved by the FDA in 2016 for the
18
management of pain and inflammation associated with osteoarthritis in dogs. Another
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound, BGC20-1531 (3), discovered by Pharmagene using a virtual screening
method, was advanced to Phase II clinical development for alleviating the symptoms of
19
migraine headache. In 2015, Eisai unveiled its EP4 antagonist E7046 (4), which was
capable of preventing the differentiation of monocytic myeloid lineage cells into a pro-
tumorigenic phenotype in the tumor microenvironment and was advanced to phase I
20
clinical trial in patients with diverse cancer types. Despite their chemical structures being
21
22
not available yet, some EP4 antagonists such as ONO-4578 and LY3127760 are now
under development at different stages of clinical studies including advanced or metastatic
solid tumors and rheumatoid arthritis. In addition, some novel preclinical EP4 antagonists,
23
24
25
26
as exemplified by CJ-042794 (5) , MF-2894 (6) , MF-766 (7) and BAY 1316957 (8)
can also serve as chemical tools in studying the EP4 receptor and its role in tumors.
ACS Paragon Plus Environment
6

Page 7 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
F
O
F
O
N
N
OH
N
H
OH
O
O
O
O
N
O
O
O
N
O
S
N
S
O
N
HN
N
HO
O
H
Na+
O
HO
CF3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
PGE2
Grapiprant (CJ-023,423)
BGC20-1531 (AP-1531)
E7046
O
O
O
O
Cl
N
N
H
N
H
H
S
N
N
OH
OH
OH
O
N
N
OH
O
O
O
O
CF3
F
5
CF3
6
7
8
CJ-042794
MK-2894
MF-766
BAY 1316957
Figure 1. The Chemical Structure of PGE and Selected EP4 Antagonists.
2
On the basis of chemical structure features, the present EP4 antagonists can be
roughly grouped into two broad categories: the first-generation EP4 antagonists
containing sulfonyl urea/acylsulfonamide moieties, and the second-generation ligands
bearing carboxamido-benzoic acid and related scaffolds.27, 28 With the exception of
compound 2, the development of the first-generation EP4 antagonists for clinical use was
unsuccessful primarily due to their poor pharmacokinetic profiles such as species-
dependent metabolic liability and low blood-brain barrier permeability.24, 25, 29 Therefore,
we turned our attention to the second-generation EP4 antagonists reported in literature
which exhibited better pharmacokinetic and metabolism profiles while maintaining high
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency and selectivity towards the EP4 receptor. Through structure-activity relationship
(SAR) analysis, it was found that these second-generation EP4 antagonists generally
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
included three essential structural requirements: a core skeleton, a benzoic-acid-
containing side chain, and a hydrophobic side chain (Figure 2). The chemical structure of
the benzoic-acid-containing side chain was relatively conserved, and it was located
adjacent to the hydrophobic side chain in the core skeleton. To date, multiple structurally
distinct scaffolds have been introduced into the core skeleton such as phenyl ring,
thiophene, indole, and pyrazole as outlined in Figure 1. It is noteworthy that most of the
second-generation EP4 antagonists reported in the literature have relatively limited
30
31
24
25
structural modifications on their core scaffolds, such as compound 4 , 5 , 6 , and 7 .
In the process of our efforts to identify novel EP4 antagonist, 1,2,3-triazole drew our
attention. The 1,2,3-trizole ring is present in many biologically active compounds including
several marketed drugs (Tazobactam, Rufinamide, Suvorexant, Cefatrizine), and is one
of the of privileged scaffolds in medicinal chemistry possessing unique structural
features.32 The unique structural characteristics of 1,2,3-triazole like strong dipole
moments, multiple hydrogen bond receptors and marked stability under metabolic
ACS Paragon Plus Environment
8

Page 9 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
conditions make it an ideal bioisostere for various functional groups to improve molecule
drug-like properties, including bioavailability and water solubility.33 In addition, the
synthetic routes of triazole derivatives are feasible and reliable, which could facilitate the
follow-up lead optimization process. We assume that incorporation of a 1,2,3-triazole ring
as core skeleton would be a reasonable strategy to develop novel EP4 antagonists. Some
of the compounds reported in this work exhibited potent EP4 receptor antagonistic activity
and good selectivity over other EP receptor subtypes. Cytotoxicity and preliminary
ADMET studies were further carried out to evaluate their drug-like property. At last,
immunological effects in vitro and anti-tumor efficiency were also assessed for the best
compound 59 in this series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. An overview of the design and optimization of triazole derivatives reported in this
study.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
Chemistry. The syntheses of triazole analogs 15 - 49 is described in Scheme 1. The
bromide starting material 9 was treated with NaN to afford the corresponding azide 10,
3
which underwent a traditional 1,3-dipolar cycloaddition reaction with different alkynoates
to simultaneously yield a mixture of two triazole regioisomers.34 After chromatographic
separation, the structures of isomers 11 and 12 were determined by X-ray crystallography
or 2D NMR spectroscopy (see Supporting Information for more details). It was interesting
to note that the 1,4-regioisomer 11 had a lower R value than its 1,5-counterpart 12 when
f
monitored by thin-layer chromatography (TLC) analysis and this phenomenon was
applicable to all of the triazole intermediates synthesized in this work. After hydrolysis of
the ester group, compound 11 was subjected to HATU coupling reaction with methyl-4-
[
(1S)-1-aminoethyl]benzoate and subsequent benzoate hydrolysis to obtain the desired
ACS Paragon Plus Environment
10

Page 11 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
products 15 - 24. Compounds 25 - 49 were prepared from 12 by the same sequence of
steps.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthetic routes of compounds 50 - 65 were outlined in Scheme 2. 1-
(Bromomethyl)-4-(trifluoromethyl)benzene 9b was initially converted to the azide and then
reacted with ethyl 3-bromopropiolate to form 1,5-regioisomer 13. Subsequent Suzuki
cross-coupling with substituted boric acids or Stille cross-coupling with organostannane
yielded esters 14c-j and 14k-l, respectively. In addition, esters 14a-b were prepared via
Pd/C-catalyzed hydrogenation of cycloalkenyl analogs 14c-d. Compounds 50 - 65 were
synthesized from 14a-j in the same sequence of steps as compounds 25 - 49.
Scheme 1. Synthesis of Triazole Analogs 15 - 49a
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
4
O
OEt
R2
N
N
(S)
N
N
5
c, d, c
N
H
N
N 1
R1
R2
COOH
COOH
R1
R1 Br
a
R1 N3
b
11a-j
15 - 24
R2
R2
9
10
4
N
9
9
9
9
9
9
9
9
9
a R1 = 4-F-Ph-
N
5
OEt
c, d, c
H
(S)
N
b R1 = 4-CF3-Ph-
N
N
N
N 1
R1
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c R1 = 3-CF3-Ph-
R1
O
1
d R = 4-F-Ph-CH2-
e R1 = 4-F-Ph-OCH2-
12a-y
25 - 49
12o R1 = 4-MeO-Ph-, R2 = Ph
f R1 = Ph-
12p R1 = 3-MeO-Ph-, R2 = Ph
g R1 = 3,4-diF-Ph-
12q R1 = 3-F-4-MeO-Ph-, R2 = Ph
12r R1 = 3,4-diMeO-Ph-, R2 = Ph
h R1 = 4-MeO-Ph-
i R1 = 3-MeO-Ph-
11a-12a R1 = 4-F-Ph-, R2 = CF3
11b-12b R1 = 4-CF3-Ph-, R2 = CF3
11c-12c R1 = 3-CF3-Ph-, R2 = CF3
11d-12d R1 = 4-F-Ph-CH2-, R2 = CF3
11e-12e R1 = 4-F-Ph-OCH2-, R2 = CF3
11f-12f R1 = 4-CF3-Ph-, R2 = H
11g-12g R1 = 4-CF3-Ph-, R2 = Me
11h-12h R1 = 4-CF3-Ph-, R2 = Et
12s R1 = 4-F-Ph-OCH -, R2 = Ph
2
9j R1 = 3-F-4-MeO-Ph-
11i-12i R1 = 4-CF3-Ph-, R2 = n-Pr
12t R1 = 4-CF -Ph-OCH -, R = Ph
2
3
2
9
k R1 = 3,4-diMeO-Ph-
11j-12j R1 = 4-CF3-Ph-, R2 = Ph
12u R1 = 3-CF -Ph-OCH -, R = Ph
2
3
2
l R1 = 4-CF3-Ph-OCH2-
12k R1 = Ph-, R2 = Ph
1
2
9
1
2
v
R
=
t
e
t
r
a
h
y
d
r
o
-
2
H
-
p
y
r
a
n
-
4
-
y
l
,
R
=
P
h
9m R1 = 3-CF3-Ph-OCH2-
12l R1 = 4-F-Ph-, R2 = Ph
12w R1 = cyclohexyl, R2 = Ph
9
9
n R1 = tetrahydro-2H-pyran-4-yl
12m R1 = 3,4-diF-Ph-, R2 = Ph
12x R1 = 4-CF -Ph-, R2 = cyclopropyl
3
o R1 = cyclohexyl
12n R1 = 3-CF3-Ph-, R2 = Ph
12y R1 = 4-CF -Ph-, R2 = cyclobutyl
3
a
Reagents and conditions: (a) NaN , dimethyl sulfoxide (DMSO), room temperature (rt),
3
overnight, 90-100%; (b) substituted or unsubstituted ethyl propiolates, toluene, reflux,
overnight, 11a-j, 29-42%, 12a-y, 20-45%; (c) LiOH·H O, MeOH/THF/H O = 2:2:1, reflux,
2
2
2 h, 92-100%; (d) methyl-4-[(1S)-1-aminoethyl]benzoate, 2-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine
(DIPEA), rt, overnight, 65-80%.
Scheme 2. Synthesis of Triazole Analogs 50 - 65a
ACS Paragon Plus Environment
12

Page 13 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R2
O
COOH
OEt
R2
N
1
1
4a R2 = cyclopentyl
H
f, g, f
N
4b R2 = cyclohexyl
N
(S)
N
N
N
N
O
F3C
F3C
50 - 51
1
4a-b
e
R2
COOH 14c R2 = cyclopent-1-en-1-yl
4d R2 = cyclohex-1-en-1-yl
14e R2 = 3,6-dihydro-2H-pyran-4-yl
O
OEt
R2
N
1
H
N
N
(S)
c
f, g, f
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4f R2 = 4-fluorophenyl
1
Br
O
O
OEt
Br
N
N
14g R2 = thiophen-3-yl
F3C
F3C
52 - 59
1
4c-j
14h R2 = furan-3-yl
N
a, b
1
4i R2 = 5-methylthiophen-2-yl
N
R2
N
O
F3C
OEt
R2
COOH 14j R2 = prop-1-en-1-yl
R2 = allyl
14l R2 = prop-1-yn-1-yl
N
CF3
N
H
(S)
d
f, g, f
N
14k
9
b
13
N
N
N
O
F3C
OEt
F3C
60 - 61
1
4k-l
R3
O
O
NH
N
N
N
N
N
N
F3C
F3C
14j
62 - 65
a
Reagents and conditions: (a) NaN , DMSO, rt, overnight, 99%; (b) ethyl 3-
3
2
bromopropiolate, toluene, reflux, overnight, 28%; (c) R -B(OH) , Pd(PPh ) ,
2
3 4
toluene/EtOH/1N Na CO = 2/1/2, 70 °C, 10 h, 32-64%; (d) allyltributylstannane or
2
3
tributyl(prop-1-yn-1-yl)stannane, 10 mol% Pd(OAc) , PPh , DMF, 95 °C, 12 h, 60-64%;
2
3
(
e) Pd/C, H , EtOH, rt, 12 h, 100%; (f) LiOH·H O, MeOH/THF/H O = 2:2:1, reflux, 2 h, 90-
2
2
2
1
00%; (g) methyl-4-[(1S)-1-aminoethyl]benzoate, HATU, DIPEA, rt, overnight, 65-83%.
In Vitro Functional Studies. The calcium flux assay is an in vitro cell-based functional
assay for rapidly screening GPCR ligands.35, 36 To assess the antagonistic activity of
these triazole analogs at the EP4 receptor, we established a calcium flux assay by co-
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
expressing G and human EP4 receptor in CHO cells. All synthetic compounds were
16
initially evaluated for their inhibitory activity at 10 M in the presence of 10 nM PGE2.
Subsequently, those compounds exhibiting more than 50% inhibition were further
evaluated for their IC50 values. Compound 4 was simultaneously tested as an active
comparator and its antagonism activity (human EP4: IC = 7.5 ± 0.6 nM, mouse EP4:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
IC = 181.1 ± 14.6 nM) was consistent with previously reported data (human EP4: IC
50
50
37
=
13.5 nM, mouse EP4: K = 371 nM). Initial exploration of the structure-activity
i
relationships (SARs) between the 1,4- and 1,5-regioisomers began with a brief
2
examination of R groups. As shown in Table 1, the antagonism activity of 1,5-regioisomer
2
triazoles (compounds 30, 31, 26, and 32 - 34) improved as the size of R substituent
increased, suggesting that large steric hindrance was preferred in this position. On the
other hand, the IC50 values of their counterparts (compounds 20, 21, 16, and 22 - 24)
were more than 10 M, indicating that the 1,5-regioisomer triazole was favorable for
maintaining potency. It was noteworthy that the antagonism activity could be influenced
dramatically by the substitution pattern of the benzyl group in compound 26. Shifting the
trifluoromethyl group from the para-position to meta-position (compound 27) or replacing
ACS Paragon Plus Environment
14

Page 15 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the trifluoromethyl group with a fluorine atom (compound 25) caused a 10-fold decrease
in antagonistic activity at the EP4 receptor when compared to compound 26. The insertion
of an extra carbon atom between the benzyl group and the triazole core in compound 25
led to complete loss of antagonistic activity at the EP4 receptor (compound 28) while
compound 29 with one more oxygen atom maintained the activity. Since compound 34
was the best ligand so far with single-digit nanomolar potency, further optimization efforts
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
around its R substituent were then carried out. As shown in Table 2, a phenyl ring was
essential to maintain the potency as tetrahydropyran 46 and cyclohexane 47 completely
lost their activity against the EP4 receptor. The introduction of various substituents into
the phenyl ring increased the potency of triazole analogs to some extent, especially those
ligands possessing a trifluoromethyl or methoxyl group at the para-position (34 and 39).
Although extending the distance between the phenyl ring and the triazole core (43 - 45)
was beneficial for the potency, compound 45 was readily degraded in human/mouse liver
microsome stability assays indicating that these ligands did not deserve further
consideration.
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Antagonistic Effect of Compounds 15 - 34 against the Human EP4 Receptora
R1
R2
O
O
N
N (S)
N (S)
N
N
N
N
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
N
COOH
R2
COOH
1,4-regioisomer
1,5-regioisomer
R1
R2
b
R1
R2
b
Compd.
15
IC (nM)
Compd.
25
IC (nM)
50
50
2315.7 ±
53.8
224.8 ± 2.9
CF3
CF3
CF3
>104
>104
>104
CF3
CF3
CF3
CF3
F
F
2
16
17
18
26
27
F3C
F3C
1970.0 ±
163.2
CF3
CF3
F
F
CF3
CF3
H
>104
>104
>104
28
29
30
31
32
33
34
>104
O
O
19
20
21
22
CF3
275.5 ± 1.3
>104
F
F
H
Me
Et
F3C
F3C
F3C
F3C
F3C
F3C
F3C
F3C
F3C
F3C
Me
Et
>104
>104
>104
>104
23
n-Pr
Ph
>104
>104
n-Pr
Ph
68.8 ± 3.0
8.5 ± 3.0
24
aSee Experimental Section. Data represent mean values ± SEM of at least three independent calcium
b
flux experiments using CHO-human EP4-G . The IC values represent the antagonistic activity at
16
50
the human EP4 receptor. Compound 4 was used as an active comparator with IC50 value of 7.5 ± 0.6
nM against the human EP4 receptor.
ACS Paragon Plus Environment
16

Page 17 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
In an attempt to further improve its EP4 potency, the R substituent of compound 34
was again interrogated. Replacing the phenyl ring in compound 34 with a series of
cycloalkyl groups yielded triazole analogs (48 - 54) that were approximately 3- to 20-fold
less potent than parent compound 34. In contrast, heterocyclic analogs (55 - 58)
maintained the EP4 activity, especially compound 58 which was equipotent to 34.
Interestingly, incorporation of a less lipophilic allylic or propargyl group produced
compounds 59 and 61 that maintained the EP4 potency relative to compound 34, while
terminal double bond analog 60 demonstrated a significant loss in potency, suggesting
that a conjugated system composed of an unsaturated bond linked to the triazole ring
was generally beneficial for EP4 activity. Along with above-mentioned SAR results, the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
R substituents in the triazole core had an important impact on the synthesized
compounds’ activity. At last, some modifications of the (S)-4-(1-aminoethyl)benzoic acid
group in compound 59 were found to be tolerated and resulted in analogs with similar
potency compared to the parent compound, with the exception of compound 63 bearing
a (R)-methyl group opposite to that of compound 59.
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Antagonistic Effect of Compounds 35 - 47 against the Human EP4 Receptora
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
H
N
N
COOH
R1
Compd.
35
R1
IC (nM)
b
5
0
646.6 ± 207.0
330.2 ± 106.2
147.5 ± 46.9
36
F
F
37
38
39
40
F
23.0 ± 7.5
9.3 ± 2.9
CF3
O
96.8 ± 30.7
O
F
41
29.8 ± 10.7
O
O
4
2
3
119.4 ± 40.3
O
O
4
3.1 ± 0.1
F
O
44
49.1 ± 6.4
F3C
O
45
46
1.3 ± 0.1
CF3
O
>104
>104
47
aSee Experimental Section. Data represent mean values ± SEM of at least three independent calcium
b
flux experiments using CHO-human EP4-G . The IC values represent the antagonistic activity at
16
50
ACS Paragon Plus Environment
18

Page 19 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the human EP4 receptor. Compound 4 was used as an active comparator with IC50 value of 7.5 ± 0.6
nM against the human EP4 receptor.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Antagonistic Effect of Compounds 48 - 65 against the Human EP4 Receptora
R2
N
H
N
N
R3
N
O
F3C
b
Compd.
R2
R3
IC (nM)
5
0
4
8
9
52.1 ± 4.1
75.3 ± 4.2
65.1 ± 4.7
161.3 ± 10.5
27.9 ± 2.7
183.0 ± 16.0
118.8 ± 19.9
57.9 ± 5.8
12.9 ± 1.3
45.6 ± 4.7
4.6 ± 0.1
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
4
50
51
52
53
54
55
O
F
S
56
57
O
58
S
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6
6
9
0
1
6.1 ± 0.2
COOH
>103
32.0 ± 1.5
13.9 ± 1.4
>103
COOH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
COOH
COOH
62
63
COOH
COOH
COOH
6
4
5
10.5 ± 0.5
13.9 ± 1.1
6
aSee Experimental Section. Data represent mean values ± SEM of at least three independent calcium
b
flux experiments using CHO-human EP4-G . The IC values represent the antagonistic activity at
16
50
the human EP4 receptor. Compound 4 was used as an active comparator with IC50 value of 7.5 ± 0.6
nM against the human EP4 receptor.
Next, selected triazole analogs with the IC value below 50 nM at the human EP4
50
receptor were further tested for their inhibitory potency at the mouse EP4 receptor and
selectivity over human EP1-3 receptors (Table 4). As shown in Table 4, most of these
compounds exhibited moderate to potent antagonistic activity at the mouse EP4 receptor.
In general, compounds bearing an unsaturated alkyl chain or a heterocyclic ring in the 4-
position of the 1H-1,2,3-triazole core showed better activity than cycloalkane and phenyl
ACS Paragon Plus Environment
20

Page 21 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
analogs, especially compound 59 exhibiting an IC50 value below 20 nM. As for the
selectivity, most of the tested compounds had no significant interactions with human EP1-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
receptors at concentrations up to 10 M, with the exception of compound 61 which had
very weak inhibitory activity at the human EP3 receptor.
Table 4. Antagonistic Activity and Subtype Selectivity of Selected Compoundsa
Selectivity
Human EP4 Mouse EP4
Compd.
4
Human EP1 Human EP2 Human EP3
b
(
IC , nM)
(IC , nM)
50
50
(
IC , nM)
(IC , nM)
(IC , nM)
5
0
50
50
7.5 ± 0.6
8.5 ± 3.0
3.1 ± 0.1
1.3 ± 0.1
181.1 ±14.6
906.3 ± 23.2
132.3 ± 2.7
135.2 ± 1.8
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
34
43
45
52
56
57
27.9 ± 2.7 400.1 ± 23.6
12.9 ± 1.3
45.6 ± 4.7
4.6 ± 0.1
6.1 ± 0.2
27.5 ± 1.9
57.6 ± 3.0
42.1 ± 2.9
16.2 ± 1.7
58
59
2
922.3 ±
>104
>104
6
1
32.0 ± 1.5
13.9 ± 1.4
70.9 ± 6.3
33.7 ± 5.0
2
76.4
ND
ND
ND
62
NDc
ND
ND
ND
ND
ND
6
4
5
10.5 ± 0.5 130.0 ± 10.9
13.9 ± 1.1 219.7 ± 23.3
6
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ɑ
See Experimental Section. Data represent mean values ± SEM of at least three independent
b
experiments determinations via calcium flux assay using CHO-EP1-4-G . TheIC values represent
16
50
c
the antagonistic activity at EP1-4 receptor. ND: not determined.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Preliminary ADME evaluation. Encouraged by the favorable biological data in vitro, we
submitted selected triazole analogs for preliminary ADME tests. When incubated with
human or pooled CD1 mouse liver microsomes,38 at least 65% of tested compounds
remained unchanged after 1h incubation at 10 M, with the exception of the short half-
time of compound 45 (t1/2 < 40 min).
Table 5. Human or Mouse Liver Microsome Stability Data for Selected Compounds
HLM
CLint(mic)b
MLM
CLint(mic)
t1/2ɑ
Remaining
t1/2
Remaining
Compd.
(min) (L/min/mg) (T=60 min) (min) (L/min/mg) (T = 60 min)
3
4
>145
31.4
<9.6
44.2
<9.6
<9.6
<9.6
<9.6
<9.6
71.3
130.6
84.9%
25.2%
90.2%
105.8%
87.9%
100.2%
89.8%
0.0%
NDc
38.4
ND
ND
36.1
ND
ND
28.5%
ND
45
52
56
57
>145
>145
>145
>145
>145
>145
ND
<9.6
ND
75.5%
ND
59
>145
ND
<9.6
ND
82.0%
ND
61
testosterone 19.4
diclofenac 10.6
2.9
478.8
25.8
0.0%
44.1%
1.9%
53.6
ACS Paragon Plus Environment
22

Page 23 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
propafenon
e
6
.3
219.6
0.2%
1.2
1145.8
0.1%
ɑt1/2 is half-life. CL
b
is the intrinsic clearance; CLint(mic) = 0.693 /half-life /mg microsome protein per
int(mic)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
mL. ND: not determined.
The inhibitory effect of compounds 45, 56, 59, 61, and 4 on in vitro cytochrome P450
(CYP) activity in human liver microsome was screened using a high-throughput multiple
CYP assay for CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4. At a
concentration of 20 M, tested compounds moderately inhibited CYP2C9 and CYP2E1,
but had only weak (< 40%) inhibition of CYP2D6 and CYP3A4, two major CYP isomers
39
involved in the metabolism of about 40% of marketed drugs. Overall, compounds 56
and 59 displayed the best profiles in assays for CYP inhibition.
Table 6. CYP Inhibition Data for Selected Compounds (20 M)
Compd.
CYP1A2
30%
CYP2B6
51%
CYP2C9
67%
CYP2D6
30%
CYP2E1
67%
CYP3A4
NAɑ
45
56
21%
28%
48%
25%
63%
NA
NA
NA
NA
5
9
1
27%
26%
33%
23%
66%
6
82%
34%
69%
38%
76%
4
41%
23%
67%
35%
79%
ɑNA: not active, defined as no inhibitory activity.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The pharmacokinetics (PK) profiles of selected triazole ligands were further characterized
in BALB/c mice (Table 7).40 When intravenously administered at a dose of 1 mg/kg,
compounds 56 and 59 demonstrated moderate clearance (56: CL = 3.0 L/h/kg, 59: CL =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.7 L/h/kg) in mice with a corresponding favorable half-life of 3.3 h and 4.1 h, respectively.
Both of them exhibited reasonable oral exposures (56: AUC0-ꝏ = 654.2 ng·h/mL, 59:
AUC0-ꝏ = 1243.5 ng·h/mL) and good bioavailability (56: F = 40.9%, 59: F = 48.0%) when
dosed orally (5 mg/kg). In general, the PK parameters of compound 59 featuring an allylic
moiety was superior to that of thiophene analog 56.
Table 7. Pharmacokinetic Parameters of Compounds 56 and 59 in BALB/c Mice
Dose
mg/kg
)
Vz
Compd Rout
Cmax
AUC0-ꝏ
Vss
CL
t1/2
F
(
(L/kg)
.
e
(ng/mL) (ng·h/mL) (L/kg)
(L/h/kg) (h) (%)
3
.
iv
1
5
1
5
808.1
365.0
1195.8
194.4
331.6
654.2
574.8
1243.5
4.0
4.4
14.5
10.2
3.0
1.7
3
56
4.
40.
9
po
iv
6
4.
1
59
4. 48.
po
7
0
ACS Paragon Plus Environment
24

Page 25 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 59 inhibited EP4-mediated cAMP signaling pathway activation. Given its good
potency and selectivity against the human EP4 receptor as well as its favorable ADME
properties, compound 59 was selected for further profiling. A number of studies have
reported that EP4 receptor stimulation can raise intracellular cAMP levels through the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9, 28
The GlosensorTM cAMP assay is an extremely sensitive
activation of the G pathway.
S
and easy-to-use luciferase biosensor-based assay enabling facile measurements of
41
intracellular cAMP accumulation in living cells. To evaluate the effect of compound 59
on intracellular cAMP accumulation, we co-expressed EP4 and pGloSensor™-22F cAMP
plasmids in HEK293 cells. As shown in Figure 3A, compound 59 dose-dependently
inhibited PGE -stimulated cAMP accumulation in HEK293-EP4 cells, with an IC value
2
50
of 18.7 ± 0.6 nM. The cAMP-response element (CRE) binding reporter assay is another
commonly used method for GPCR ligand evaluation.42, 43 We found that compound 59
dose-dependently inhibited the activity of the CRE reporter in HEK293 cells co-
transfected CRE-luc plasmids, with an IC value of 5.2 ± 0.4 nM (Figure 3C). Compound
50
4
served as an active comparator in the Glosensor and CRE reporter assays (Figure 3B,
3D). Activation of EP4 receptor can also stimulate G protein-independent -arrestin
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
signaling pathway7, 44. We next sought to investigate whether compound 59 was able to
®
45
affect -arrestin-dependent signaling by using a NanoBiT -arrestin recruitment assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The results shown that compound 59 dose-dependently inhibited PGE -stimulated -
2
arrestin recruitment in HEK293-EP4 cells, with an IC value of 0.4 ± 0.1 nM, which was
50
about 5-fold more potent than that of compound 4 (Figure S2). Additionally, considerable
evidence has recently indicated that the extracellular signal-regulated kinase (ERK) was
46
one of the key cellular effectors activated by GPCRs. To assess the effect of compound
59 on intracellular ERK activation, CHO-EP4 cells were pretreated with indicated
concertation of compound 59 and then subjected to PGE (30 nM) stimulation. Notably,
2
compound 59 reversed PGE -induced ERK phosphorylation in a concentration-
2
dependent manner (Figure 3E). Furthermore, it was found that increasing the
concentration of compound 59 shifted the PGE response curve rightwards without
2
changing the maximal response of cAMP in Glosensor cAMP assay (Figure 3F). The pA2
value calculated from the Schild plot analysis was 8.25 with a slope of 1.00 (Figure 3G).
Compound 59 had a more than 1000-fold selectivity over EP1, EP2, and EP3 receptors
ACS Paragon Plus Environment
26

Page 27 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
Figure 3H), suggesting that compound 59 was a highly potent and selective competitive
EP4 antagonist.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The antagonistic effects of compound 59 on human EP4. (A-B) Effect of compound 59 and
4
on PGE -induced intracellular cAMP accumulation determined by the Glosensor cAMP assay. (C-
2
D) Dose-response curve of compound 59 and 4 in CRE reporter assay. (E) Western blot analysis of
phosphorylated ERK1/2 in CHO-EP4 cells. CHO-EP4 cells were pretreated with indicated compounds
for 20 min and then subjected to 30 nM PGE simulation for 10 min. Compound 4 (10 M) was used
2
as an active comparator. (F) Schild plot analyses of compound 59 by Glosensor cAMP assay. (G) The
pA and slope of this Schild plot (F) were 8.25 and 1.00, respectively. (H) Dose-response curve of
2
compound 59 against human EP1-4 receptors in the calcium flux assay. Data were normalized as
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 118
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
percentage of maximum response, and presented as mean ± SEM of three independent experiments.
n = 3 per group.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 59 reduced tumor immunosuppression-related gene expression in Raw264.7
cells. Previous studies have demonstrated that PGE /EP4 pathway activation could
2
reprogram the tumor microenvironment through regulating the expression of a series of
immuno-related genes, which consequently suppressed cytotoxic T cell-mediated anti-
tumor immunity.47, 48 Inflammatory factors such as IL-1, IL-4, and IL-6 can promote the
expansion of immature immunosuppressive myeloid cells.49, 50 Metabolic enzymes (ARG-
1
, iNOS and COX2), and cytokines (IL-10 and CXCL-1) in the tumor microenvironment
could block T cells-mediated tumor killing.51-53 We then sought to determine the effects of
compound 59 on the expression of tumor immunosuppression-related genes in RAW
264.7, a monocyte/macrophage-like cell line. As shown in Figure 4, stimulating RAW
2
64.7 cells with GM-CSF/IL-4 plus 10 nM PGE significantly induced the expression of
2
multiple immuno-related genes including IL-1 (65.7-fold, P < 0.001), IL-4Rꝏ (2.1-fold,
P < 0.01), IL-6 (2.5-fold, P < 0.001), ARG1 (73.0-fold, P < 0.001), iNOS, COX2 (4.3-fold,
ACS Paragon Plus Environment
28

Page 29 of 118
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
P < 0.01), IL-10 (2.3-fold, P < 0.05) and CXCL1 (1.9-fold, P < 0.001). Notably, compound
59 dose-dependently inhibited the expression of these immuno-related genes.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Collectively, these findings revealed that the EP4 blockade by compound 59 remarkably
suppressed the expression of tumor immunosuppression-related genes.
Figure 4. Compound 59 repressed the expression of immuno-related genes in Raw 264.7 cells. Raw
2
64.7 cells were treated with GM-CSF/ IL-4/ PGE ± 4 (10 M) or 59 (0.1 M, 1 M, 10 M) for 24 h.
2
The mRNA expression of IL-1 (A), IL-4R (B), IL-6 (C), ARG1 (D), iNOS (E), COX2 (F), IL-10 (G)
ACS Paragon Plus Environment
29